CN101585877B - Method for synthesizing rhodamine B complete antigen - Google Patents
Method for synthesizing rhodamine B complete antigen Download PDFInfo
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- CN101585877B CN101585877B CN2009100317275A CN200910031727A CN101585877B CN 101585877 B CN101585877 B CN 101585877B CN 2009100317275 A CN2009100317275 A CN 2009100317275A CN 200910031727 A CN200910031727 A CN 200910031727A CN 101585877 B CN101585877 B CN 101585877B
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- rhodamine
- complete antigen
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Abstract
The invention discloses a method for synthesizing rhodamine B complete antigen, and belongs to the technical field of biochemical engineering. The method takes rhodamine B as a hapten, couples the rhodamine B with carrier protein BSA by a mixed acid anhydride method, and measures the coupling ratio of a conjugate by a spectrophotometry. The method successfully synthesizes the complete antigen of rhodamine B, has simple and effective synthesis steps, can be fully used in immunoassay, provides necessary artificial antigen for future research of people, and can meet the requirement of national rhodamine B research.
Description
Technical field
A kind of synthetic method of rhodamine B complete antigen belongs to technical field of biochemical industry.
Background technology
Rhodamine B, English name: Rhodamine B; CAS:81-88-9, molecular formula C
28H
31ClN
2O
3Rhodamine B is a kind of dyestuff with bright pinkish synthetic, and intensive fluorescence is arranged in solution, as industries such as cell fluorescence staining agent, tinted shade, characteristic fireworks and firecrackers in the laboratory.It is the triphenylmethane basic dyestuff, has the carcinogenic and mutagenicity of potential, and China and European Union etc. does not allow to use in food.Rhodamine B has fat-soluble, is used as seasonings (mainly being red chilly powder and paprika oleoresin) staining agent.Can cause residual when having used the food that contaminated seasonings makes.Content was higher when seasonings used rhodamine B dyeing, even directly mixed, and can carry out the scene and detect.Because of its content is lower, need send the laboratory to detect in the food.The food classification that may add is a seasonings.Existing instrument detecting method is difficult to realize rhodamine B is carried out quick, accurate, how residual detection.At home and abroad there is no the report of ideal at present at the how residual immunologic detection method of rhodamine B.In order to remedy this blank, designed with rhodamine B from complete antigen as the bright B immunodetection of haptenic synthesizing rhodamine.
Summary of the invention
The synthetic method that the purpose of this invention is to provide a kind of rhodamine B complete antigen.Prepared product is used for the immune analysis method research of rhodamine B.For people's research from now on provides essential artificial antigen.
Technical scheme of the present invention: a kind of synthetic method of rhodamine B complete antigen is a haptens with the rhodamine B, with mixed anhydride method with itself and carrier proteins BSA coupling, with the coupling ratio of spectrophotometry conjugate; Step is:
(1) rhodamine B complete antigen is synthetic:
Preparation A liquid: the 20mg rhodamine B is dissolved among the 1mL DMF, adds 10 μ L isobutyl chlorocarbonate magnetic agitation 1h again after adding 10 μ L, three fourth ammonia mixings under the ice bath of complete molten back;
Preparation B liquid: 40mg BSA is dissolved in the phosphate buffered saline buffer of 3mL pH 7.4, and 4 ℃ of preservations are standby;
Under the ice bath A liquid is dropwise added in the B liquid, put 4 ℃ of following temperature then and incubate 3h, promptly get the rhodamine B complete antigen mixed solution;
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, in boiling water, boil 5min, use 60 ℃ deionized water rinsing 3min again, be kept in 4 ℃ of deionized waters standby;
Dialysis: the rhodamine B complete antigen mixed solution is moved in the dialysis tubing, with the deionized water dialysis of the phosphate buffer soln of the pH7.4 of the 0.01M of 2 * 2L and 2 * 2L 3 days, use lyophilization that the liquid in the dialysis tubing is made powder at last, promptly obtain rhodamine B complete antigen;
(2) evaluation of rhodamine B complete antigen: rhodamine B complete antigen adopts spectrophotometry to identify its coupling result, utilize the bovine serum albumin reference liquid to obtain typical curve, obtain the protein concentration of antigenic solution from the curve comparison, calculate molar absorptivity ε, measure the coupling ratio of rhodamine B complete antigen again.
Coupling ratio is measured: be by the method for the ratio of two kinds of molecules of link coupled (coupling ratio) in the estimation conjugate, though the measuring method kind is a lot, all be to be set up by the principle of two kinds of molecule contents of link coupled (or relative content) according to detecting in the conjugate.Spectrophotometry is to utilize material that the principle that absorption and its concentration of light is proportionlity is measured respectively by two kinds of molecular conecentrations of link coupled. in macromole and small molecules conjugate, two kinds of molecules all have different separately ultraviolet scanning spectrums, and show the character of spectrogram superposition.
The conjugate determination of protein concentration: compound concentration is 0,40,60,80,100 μ gmL
-1Bovine serum albumen solution 1.5mL, add 5mL coomassie brilliant blue staining liquid, mixing immediately, warm 5 minutes of 30 ℃ of water-baths, each concentration is made parallel sample, surveys light absorption value, the relation curve of drafting protein concentration and light absorption value at 595nm place.Antigenic solution is diluted by a certain percentage, measure the light absorption value of antigenic solution at the 595nm place, obtain the corresponding proteins concentration of antigenic solution from curve.
Molar absorptivity ε: preparation rhodamine B concentration is 0,10,20,30 μ gmL
-120% ethanolic soln, by UV scanning as can be known the maximum absorption wavelength of rhodamine B be 564nm, survey light absorption value at the 564nm place, each concentration is made parallel sample. molar absorptivity is calculated as: ε=light absorption value/volumetric molar concentration.
Coupling ratio is measured: prepare 150 μ gmL
-120% ethanolic soln of bovine serum albumin, with the coupled product complete antigen with 20% alcohol dilution to 150 μ gmL
-1, survey light absorption value at the 312nm place, be blank with 20% ethanol, the light absorption value of measuring bovine serum albumen solution is A1, and the light absorption value of coupled product is A2, and then coupling ratio r is: γ=[(A
1-A
2)/ε]/(150 * 10
-3/ 66200), wherein ε is molar absorptivity (Lmol
-1), 66200 is the molecular weight of bovine serum albumin, 150 * 10
-3Be bovine serum albumin concentration (μ gmL
-1).
Beneficial effect of the present invention: the present invention successfully synthesizes the artificial antigen of rhodamine B, and synthesis step is succinct, effectively, can be used for fully in the middle of the immunoassay, for people's research later on provides approach easily, can satisfy domestic needs to its research.
Description of drawings
UV scanning figure before and after the preparation of Fig. 1 rhodamine B artificial antigen.
Embodiment:
Embodiment 1
(1) preparation of rhodamine B complete antigen
Preparation A liquid: the 20mg rhodamine B is dissolved in 1mL DMF, adds 10 μ L isobutyl chlorocarbonate magnetic agitation 1h again after adding 10 μ L, three fourth ammonia mixings under the ice bath of complete molten back.
Preparation B liquid: 40mg BSA is dissolved in the phosphate buffered saline buffer of 3mL pH 7.4, and 4 ℃ of preservations are standby.
Under the ice bath A liquid is dropwise added in the B liquid, put 4 ℃ of following temperature then and incubate 3h.Promptly get the rhodamine B complete antigen mixed solution.
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, in boiling water, boil 5min, use 60 ℃ deionized water rinsing 3min again, be kept in 4 ℃ of deionized waters standby.
Dialysis: the rhodamine B complete antigen mixed solution is moved in the dialysis tubing, dialysed 3 days with the PBS solution of the pH7.4 of the 0.01M of 2 * 2L and the deionized water of 2 * 2L.Use freeze-drying that the liquid in the dialysis tubing is made powder at last, promptly obtain rhodamine B complete antigen.
2) evaluation of rhodamine B artificial antigen
The conjugate determination of protein concentration: compound concentration is 0,40,60,80,100 μ gmL
-1Bovine serum albumen solution 1.5mL, add 5mL coomassie brilliant blue staining liquid, mixing immediately, warm 5 minutes of 30 ℃ of water-baths, each concentration is made parallel sample. survey light absorption value, the relation curve of drafting bovine serum albumin concentration and light absorption value at 595nm place.Antigenic solution is diluted by a certain percentage, measure the light absorption value of antigenic solution at the 595nm place, obtain the corresponding proteins concentration of antigenic solution from curve.The protein concentration that this experimental calculation gets antigenic solution is 3.28mgmL
-1
Molar absorptivity ε: preparation rhodamine B concentration is 0,10,20,30 μ gmL
-120% ethanolic soln, by UV scanning as can be known the maximum absorption wavelength of rhodamine B be 564nm, survey light absorption value at the 564nm place, each concentration is made parallel sample. molar absorptivity is calculated as: ε=light absorption value/volumetric molar concentration.This experimental calculation gets ε=7737.3Lmol
-1
Coupling ratio is measured: prepare 150 μ gmL
-120% ethanolic soln of bovine serum albumin, with coupled product with 20% alcohol dilution to 150 μ gmL
-1, survey light absorption value at the 564nm place, be blank with 20% ethanol, the light absorption value of measuring bovine serum albumen solution is A1, and the light absorption value of coupled product is A2, and the coupling ratio r is: γ=[(A
1-A
2)/ε]/(150 * 10
-3/ 66200), then coupling ratio r is: 11.67, and wherein ε is molar absorptivity (Lmol
-1), 66200 is the molecular weight of bovine serum albumin, 150 * 10
-3Be bovine serum albumin concentration (μ gmL
-1).
Claims (1)
1. the synthetic method of a rhodamine B complete antigen is characterized in that with the rhodamine B being haptens, with mixed anhydride method with itself and carrier proteins BSA coupling, with the coupling ratio of spectrophotometry conjugate; Step is:
(1) rhodamine B complete antigen is synthetic:
Preparation A liquid: the 20mg rhodamine B is dissolved among the 1mL DMF, adds 10 μ L isobutyl chlorocarbonate magnetic agitation 1h again after adding 10 μ L, three fourth ammonia mixings under the ice bath of complete molten back;
Preparation B liquid: 40mg BSA is dissolved in the phosphate buffered saline buffer of 3mL pH7.4, and 4 ℃ of preservations are standby;
Under the ice bath A liquid is dropwise added in the B liquid, put 4 ℃ of following temperature then and incubate 3h, promptly get the rhodamine B complete antigen mixed solution;
Dialysis tubing pre-treatment: get the dialysis tubing of 10cm, in boiling water, boil 5min, use 60 ℃ deionized water rinsing 3min again, be kept in 4 ℃ of deionized waters standby;
Dialysis: the rhodamine B complete antigen mixed solution is moved in the dialysis tubing, with the deionized water dialysis of the phosphate buffer soln of the pH7.4 of the 0.01M of 2 * 2L and 2 * 2L 3 days, use lyophilization that the liquid in the dialysis tubing is made powder at last, promptly obtain rhodamine B complete antigen;
(2) evaluation of rhodamine B complete antigen: rhodamine B complete antigen adopts spectrophotometry to identify its coupling result, utilize the bovine serum albumin reference liquid to obtain typical curve, obtain the protein concentration of antigenic solution from the curve comparison, calculate molar absorptivity ε, measure the coupling ratio of rhodamine B complete antigen again.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100317275A CN101585877B (en) | 2009-06-20 | 2009-06-20 | Method for synthesizing rhodamine B complete antigen |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100317275A CN101585877B (en) | 2009-06-20 | 2009-06-20 | Method for synthesizing rhodamine B complete antigen |
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| CN101585877A CN101585877A (en) | 2009-11-25 |
| CN101585877B true CN101585877B (en) | 2011-12-07 |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101871936A (en) * | 2010-06-03 | 2010-10-27 | 江南大学 | Rhodamine B ELISA detection method |
| CN103145830A (en) * | 2013-03-07 | 2013-06-12 | 南京大学 | Rhodamine B artificial antigen as well as preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101071133A (en) * | 2007-05-11 | 2007-11-14 | 江南大学 | Method for preparing nitrazepam artificial antigen |
| CN101381410A (en) * | 2008-10-23 | 2009-03-11 | 河北大学 | Rhodamine 123 artificial antigen synthesis, antibody preparation method and application |
-
2009
- 2009-06-20 CN CN2009100317275A patent/CN101585877B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101071133A (en) * | 2007-05-11 | 2007-11-14 | 江南大学 | Method for preparing nitrazepam artificial antigen |
| CN101381410A (en) * | 2008-10-23 | 2009-03-11 | 河北大学 | Rhodamine 123 artificial antigen synthesis, antibody preparation method and application |
Non-Patent Citations (3)
| Title |
|---|
| 周享春等.罗丹明B与牛血清白蛋白的相互作用研究.《化学研究与应用》.2009,第21卷(第1期),全文. * |
| 周纯等.Synthesis and Identification of Artificial Antigens and Antibodies of Three PAHs.《2007中国科协年会论文集(二)》.2007,全文. * |
| 王雪等.氯霉素全抗原的合成及鉴定.《生物技术通报》.2008,全文. * |
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| CN101585877A (en) | 2009-11-25 |
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